An alternatively spliced isoform of the ClC-3 chloride channel (ClC-3B) was recently discovered that, like CFTR, has a PDZ-binding C-terminus. Despite its predominant intracellular location when expressed heterologously, a small amount moved to the cell surface under certain conditions where it exhibited some properties similar to ORCC (outwardly rectifying chloride channels) including activation by CFTR. Because of this and because both ClC-3B and CFTR have PDZ-binding C-termini, ClC-3B is potentially of great interest to CF. We have cloned and expressed ClC-3B and the original non-PDZ binding ClC-3A. As expected as members of the ClC-3, 4, 5 subfamily of intracellular channels, both forms localized to intracellular membranes: ClC-3A to late endosomes or multivesicular bodies as found by others; and, ClC-3B to the Golgi. This Golgi localization observed with both heterologous and endogenous expression in lung epithelial cells suggested a possible relationship to the Golgi PDZ protein, GOPC (or CAL) to which CFTR also binds. ClC-3B was found to bind to GOPC, EBP-50, and PDZK1 (CAP-70). Of these, PDZK1 and GOPC promoted association between ClC-3B and CFTR. Coexpression with any of these PDZ proteins caused some relocalization of ClC-3B and GOPC over-expression accelerated turnover as it does with CFTR. In this project, we will pursue 3 specific aims with the overall goals of relocalizing ClC-3B and clarifying its relationship to CFTR. Specific Aim 1 will determine the role of GOPC and other PDZ proteins in the trafficking of ClC-3B and CFTR. Specific Aim 2 will determine how numerous consensus trafficking motifs in addition to the PDZ tail control traffic in the distal secretory path resulting in different localizations of ClC-3A, ClC-3B and CFTR. Interfering with the multiple motifs in different combinations by mutagenesis or membrane permeable peptides may enable specific channel relocalization. Specific Aim 3 will characterize the chloride channel activity of ClC-3B both in its normal location in the Golgi by fusing vesicles with planar lipid bilayers and at the cell surface by patch clamp when it has been relocalized there. Its properties with respect to voltage dependence, halide selectivity, conductance and inhibitor sensitivity will be compared with those of ORCC and the influence of co-expressed CFTR in the presence and absence of different PDZ proteins will be tested. Through these aims, this project will elucidate details of the trafficking of chloride channel proteins in the distal secretory/endocytic pathway and reveal means of their relocation.